SummaryNuclear pore complexes (NPCs) are fundamental components of all eukaryotic cells that mediate nucleocytoplasmic exchange. Elucidating their 110 MDa structure imposes a formidable challenge and requires in situ structural biology approaches. Fifteen out of about thirty nucleoporins (Nups) are structured and form the Y- and inner ring complexes. These two major scaffolding modules assemble in multiple copies into an eight-fold rotationally symmetric structure that fuses the inner and outer nuclear membranes to form a central channel of ∼60 nm in diameter 1. The scaffold is decorated with transport channel Nups that often contain phenylalanine (FG)-repeat sequences and mediate the interaction with cargo complexes. Although the architectural arrangement of parts of the Y-complex has been elucidated, it is unclear how exactly it oligomerizes in situ. Here, we combined cryo electron tomography with mass spectrometry, biochemical analysis, perturbation experiments and structural modeling to generate the most comprehensive architectural model of the NPC to date. Our data suggest previously unknown protein interfaces across Y-complexes and to inner ring complex members. We demonstrate that the higher eukaryotic transport channel Nup358 (RanBP2) has a previously unanticipated role in Y-complex oligomerization. Our findings blur the established boundaries between scaffold and transport channel Nups. We conclude that, similarly to coated vesicles, multiple copies of the same structural building block - although compositionally identical - engage in different local sets of interactions and conformations.

The nuclear pore complex (NPC) is a fundamental component of all eukaryotic cells that facilitates nucleocytoplasmic exchange of macromolecules. It is assembled from multiple copies of about 30 nucleoporins. Due to its size and complex composition, determining the structure of the NPC is an enormous challenge, and the overall architecture of the NPC scaffold remains elusive. In this study, we have used an integrated approach based on electron tomography, single-particle electron microscopy, and crosslinking mass spectrometry to determine the structure of a major scaffold motif of the human NPC, the Nup107 subcomplex, in both isolation and integrated into the NPC. We show that 32 copies of the Nup107 subcomplex assemble into two reticulated rings, one each at the cytoplasmic and nuclear face of the NPC. This arrangement may explain how changes of the diameter are realized that would accommodate transport of huge cargoes.

The stoichiometry of the human nuclear pore complex is revealed by targeted mass spectrometry and super-resolution microscopy. The analysis reveals that the composition of the nuclear pore and other nuclear protein complexes is remodeled as a function of the cell type.

Reticulons are integral membrane proteins that partition into and shape the tubular endoplasmic reticulum (ER). We propose that reticulons use a membrane insertion mechanism to generate regions of high membrane curvature in the ER. A reticulon contains two short hairpin transmembrane domains (TMDs), which could generate membrane curvature by increasing the area of the cytoplasmic leaflet. Here, we test whether the short length of these hairpin TMDs is required for reticulon membrane-shaping functions in mammalian cells. We lengthened the TMDs of reticulon 4 to resemble a typical bi-pass TMD that spans both leaflets. We find that TMD mutants oligomerize like wild type (wt), however, they are not immobilized, do not partition into tubules, do not constrict tubules and no longer suppress peripheral ER cisternae. Therefore, short hairpin TMD length is required for reticulon protein partitioning and membrane-shaping functions. Another membrane protein with a short hairpin TMD is caveolin. We show that an ER-retained caveolin construct also partitions within the ER in a manner that is dependent on it containing a short hairpin TMD. These data suggest that a short hairpin TMD may be a general feature used by membraneshaping proteins to partition into and shape regions of high membrane curvature. The endoplasmic reticulum (ER) is an essential eukaryotic organelle required for secretory and membrane protein synthesis, lipid synthesis and calcium signaling (1). The ER has an elaborate and extensive structure that includes three major domains that are easily resolved by fluorescence microscopy including (i) the nuclear envelope (NE), (ii) the peripheral ER cisternae and (iii) the tubular ER (2). The NE is composed of a double membrane bilayer that includes the inner nuclear membrane (INM) and outer nuclear membrane (ONM) (3). The INM and ONM are stacked over each other separated by a lumen called the perinuclear space (PNS) and are connected to each other at the nuclear pores. The ONM is continuous with the membrane of the peripheral ER, which is composed of the peripheral ER cisternae and peripheral ER tubules. Both the lumen and the membrane of all three of these major ER domains are continuous with each other and yet these domains have very different structures. The shape of these domains varies to a large extent because the membrane curvature of these domains varies. For example, the membrane of the NE is relatively flat and has low membrane curvature everywhere except at the nuclear pores. Likewise, the peripheral ER cisternae also have low membrane curvature except at their edges. In contrast, the tubular ER has high membrane curvature in cross-section along the length of the tubule.The various domains of the ER are likely to be structured by specific types of membrane-shaping proteins that can generate or maintain membrane curvature. It is not known how peripheral ER cisternae are shaped; it could be that the lack of membrane curvature at ER cisternae and the NE is because of the absence of proteins that generate ER...

Idiopathic pulmonary fibrosis (IPF) is the most common and the most aggressive fibrosing interstitial lung disease (ILD). Despite recent promising clinical trials, IPF remains incurable and largely untreatable. Genetic studies have identified several risk loci for both sporadic and familial forms of IPF. A single variant upstream of MUC5B is predicted to account for more than 30% of all IPF risk. This variant, rs35705950, is associated with expression of MUC5B in healthy lung tissue. However, the mechanism underlying the relationship between rs35705950 and MUC5B expression remains unclear.The first goal of my thesis research was to determine whether rs35705950 is also a risk factor for other forms of ILD. Genomic DNA from individuals with IPF, HP, COPD, iNSIP, and RB-ILD was obtained and genotyped for the common MUC5B promoter variant. In these populations the risk allele was associated with IPF and iNSIP which suggested a potential shared disease mechanism. Additionally, I showed that in the IPF and control populations rs35705950 is associated with lung MUC5B expression.The second goal of my thesis research was to investigate MUC5B promoter DNA methylation which has previously been shown to play a role in MUC5B expression.Methylation analysis of the 4KB region upstream of MUC5B identified two differentially methylated regions (DMRs) associated with IPF, one DMR associated with MUC5B iv expression, and one DMR associated with rs35705950. The IPF, MUC5B expression, and rs35705950 associated DMRs are all differentially methylated at a cluster of 11 CpG motifs. overlapping DMR, including the motif disrupted by rs35705950. These findings suggest that DNA methylation may play a role in MUC5B gene regulation and IPF risk.Next, I used cross species analysis to identify highly conserved domains within the overlapping DMR. Evolutionary conservation indicated selective pressures to maintain sequences overtime and suggests biological importance. The overlapping DMR contains a highly conserved binding motif for FOXA2, a transcription factor. Further analysis using ChIP-qPCR, reporter constructs, and siRNA, established a role for FOXA2 in regulation of MUC5B expression in lung tissue. Additionally, siRNA knock down identified 3 other transcription factors which are associated with MUC5B expression; STAT3, HOXA9 and ZBTB7A. These transcriptional regulators provide insight into possible therapeutic intervention for MUC5B dysregulation and IPF.The form and content of this abstract are approved. I recommend its publication.